A testis-specific lncRNA functions as a post-transcriptional regulator of MDM2 and stimulates apoptosis of testicular germ cell tumor cells

Germ cells preferentially induce apoptosis in response to DNA damage to avoid genomic mutations. Apoptosis of germ cells is closely related to cancer development and chemotherapy resistance; however, its regulatory mechanism is unclear. Here, we suggest that testis-specific lncRNA LINC03074 is involved in male germ cell apoptosis by regulating the expression of the proto-oncogene MDM2. LINC03074 is highly expressed in the sperm of healthy adult testes and cancer cells of testes with testicular germ cell tumors (TGCTs). LINC03074 binds to MDM2 mRNA via an Alu element, thereby reducing MDM2 protein levels. LINC03074 stimulates STAU1-mediated nuclear export of MDM2 mRNA by increasing STAU1 binding to MDM2 mRNA in the cell nucleus, thereby promoting PKR-mediated translational repression in the cytoplasm. The induction of apoptosis in TGCT cells and their responsiveness to the anticancer drug cisplatin is enhanced by LINC03074. Notably, LINC03074 increased E2F1 expression without increasing p53, the primary target of MDM2, and upregulated the apoptotic gene p73, the target gene of E2F1. LINC03074-mediated regulation of apoptosis contributes to the responsiveness of TGCTs to anticancer drug-induced DNA damage.


INTRODUCTION
Testicular germ cell tumors (TGCTs) are generally highly sensitive to platinum-based chemotherapy, such as cisplatin, but some are chemotherapy-resistant [1].Mouse double minute 2 (MDM2) is amplified in various human malignancies, including TGCTs, and MDM2 overexpression is associated with chemotherapy resistance [2,3].MDM2 is a major negative regulator of p53, promoting ubiquitin-dependent proteasomal degradation of p53 as an E3 ubiquitin ligase and repressing p53 transcriptional activation [4,5].The tumor suppressor p53 is commonly mutated in various types of cancers [6,7], whereas it is often overexpressed and rarely mutated in TGCTs [3].Accordingly, MDM2 overexpression in TGCTs is predicted to affect the activity of wild-type p53; however, the molecular mechanisms underlying the sensitivity or resistance of TGCTs to chemotherapy remain unclear.
MDM2 expression and activity are regulated at multiple levels, from transcription to numerous post-translational modifications, in addition to genomic alterations, such as copy number variations, mutations, and polymorphisms [8].Posttranscriptional regulation of MDM2 at the mRNA level has been widely reported to modulate transcript stability via miRNAs [9].Human MDM2 has a very long 3′UTR (~5.7 kb) that retains a multitude of potential miRNA targets [10].Notably, MDM2 contains multiple transposable elements containing Alu in the 3′UTR [11].Alu elements are abundant retrotransposon elements that spread throughout the human genome and occupy a significant portion of the 3′UTR [12].The inverted repeat Alu element (IRAlus) in the 3′UTR forms a double-stranded RNA (dsRNA) structure, which serves as a target for dsRNA-binding factors such as Staufen1 (STAU1) and adenosine deaminase acting on RNA (ADAR1) [13,14].STAU1 binds to IRAlu in the 3′UTR to undergo various RNA metabolic processes, such as RNA synthesis, folding, modification, processing, translation, and decay [13].ADAR1, an adenosine-to-inosine (A-to-I) RNA-editing enzyme, competes with STAU1 for the occupancy of target RNAs, thereby inhibiting STAU1-mediated nuclear retention or decay of RNAs [15,16].In MDM2 gene expression, STAU1 and ADAR1 appear to mediate post-transcriptional regulation via binding to the IRAlus of the MDM2 3′UTR, a mechanism independent of alterations in mRNA stability and miRNA targeting [16].
Here, we show the role of LINC03074, a testis-specific lncRNA with an Alu element, in TGCT cells.LINC03074 binds to MDM2 mRNA via Alu, thereby stimulating STAU1-mediated nuclear export of MDM2 mRNA.Consequently, MDM2 is reduced by PKR-mediated translational repression, which in turn promotes the apoptosis of TGCT cells.Our findings provide molecular mechanistic insights into the drug responsiveness of TGCTs by demonstrating a regulatory mechanism of apoptosis in TGCT cells.

LINC03074 shows different expression patterns between cancerous and normal sperm
To elucidate the characteristics of TGCTs, we compared the gene expression profiles of cancer tissues from seminoma patients with those of matched normal adjacent tissues [17].A total of 565 genes, among the 50,599 genes tested, exhibited a more than 2-fold increase in RNA expression in seminoma tissues compared to normal adjacent tissues, including 18 genes encoding lncRNAs (data not shown).In contrast, 431 genes exhibited a >2-fold decrease in expression in cancer tissues, including 59 genes encoding lncRNAs (data not shown).To identify the lncRNA that determines the characteristics of testicular tumor cells, we focused on LINC03074 (LOC100505685), which showed marked differences in expression between cancerous and normal tissues.According to the database, LINC03074 is expressed specifically in the testes of humans (Fig. 1A).Moreover, a recent study identified LINC03074 as a testis-specific lncRNA [18].The expression pattern of LINC03074, which was significantly higher in normal tissues than in cancerous tissues of the testes of patients with seminoma, was confirmed via relative quantitative analysis using RT-qPCR (P = 0.00178, Fig. 1B).We performed ISH with a LINC03074 detection probe in the testes of healthy adults and patients with seminomas.The results revealed that LINC03074 was localized to the nucleus and cytoplasm of normal spermatids, whereas it was mainly localized to the nucleus of seminoma cells (Fig. 1C).We further quantified the expression of LINC03074 in four types of cultured cells derived from seminoma and non-seminoma tissues and found that its expression was significantly higher in TCam-2 seminoma cells (Supplementary Fig. S1) [19].These results suggest that LINC03074 Fig. 1 Expression of LINC03074 in testis.A Expression of LINC03074 in major human tissues.The data resulted from an RNA-Seq CAGE analysis of human tissues of the RIKEN FANTOM5 project.Expression levels in tissues other than testis are less than 0.5 TPM, which is below the detection limit of the analysis.B LINC03074 levels in normal and cancer regions of testis tissues from patients with seminoma.Paired normal and cancer tissue samples were obtained from 10 different testicles (paired dots are connected by gray lines).Relative expression levels of LINC03074 to GAPDH were measured using RT-qPCR.*P = 0.00178.C In situ hybridization with detection probes for LINC03074 on testes of healthy adults and of patients with seminoma.The sense strand probe of LINC03074 was used as a negative control.The right small panels show enlarged views of the corresponding left panels.LINC03074 was localized to the nucleus and cytoplasm in normal sperm cells and mainly to the nucleus in seminoma cells.
functions in both testis-derived seminoma cells and normal cells, although it is differentially expressed in cancer and normal cells.

LINC03074 interacts with MDM2 mRNA via Alu element
To elucidate the function of LINC03074 in testicular cells, we searched databases to identify the elements with which this lncRNA could interact.Using lncRRIsearch, MDM2 mRNA was identified as a candidate interacting factor for LINC03074 [20].LINC03074 contained one Alu element, while MDM2 mRNA contained five Alu elements and a pair of inverted-Alus in the 3′ UTR (Fig. 2A).There were multiple candidate sequences for the interaction between LINC03074 and MDM2 mRNA within each Alu element (Fig. 2A).The sequences of LINC03074 and the sense strand of MDM2 mRNA were nearly complementary, as shown in an example of a candidate interaction region (ΔG = −60.91kcal/ mol, lower part of Fig. 2A).We decided to use TCam-2 cells as a model for seminoma cells in the following experiments, considering that LINC03074 is likely to function in seminomas according to its expression pattern (Fig. 1C and Supplementary Fig. S1).To determine whether LINC03074 binds to MDM2 mRNA, we performed CHART assays using TCam-2 cells.CHART enables the identification of associated targets of lncRNAs by enriching lncRNAs with their targets using affinity-tagged oligonucleotides (C-oligo) to capture endogenous lncRNAs in cross-linked cell extracts [21].LINC03074 was enriched in TCam-2 cell extracts by CHART using a C-oligo for LINC03074 (Fig. 2B).We then tested whether mRNAs of MDM2, 18S-rRNA and GAPDH were enriched using LINC03074 CHART, and found that MDM2 mRNA was associated with LINC03074 (Fig. 2B).Approximate estimates using qPCR showed that the molecular ratio of LINC03074 to MDM2 mRNA in TCam-2 cells was approximately 1:166 (Supplementary Fig. S2).To confirm whether LINC03074 and MDM2 mRNA interact via their respective Alu elements, we generated Alu elementdeficient LINC03074 and MDM2 expression constructs (Fig. 2C).We performed an RNA pull-down assay with the 3′UTR region of biotin-labeled MDM2 mRNA using total RNA extracted from HEK293 cells transiently expressing LINC03074.We found that LINC03074 (FL) binds to the MDM2 3′UTR (FL) (Fig. 2D).In comparison, LINC03074 (FL) binds to the MDM2 3′UTR (Δ5′-Alu) was attenuated (Fig. 2D).In addition, LINC03074 (ΔAlu) showed significantly weaker binding to MDM2 than LINC03074 (FL) (Fig. 2D).In contrast, no binding was detected between 18S-rRNA and MDM2 3′UTR (either FL or Δ5′-Alu) (Fig. 2D).These results indicated that LINC03074 binds to the Alu elements of MDM2 mRNA via its own Alu element.

LINC03074 inhibits MDM2 gene expression by binding to MDM2 mRNA
We investigated the effects of the interaction of LINC03074 with MDM2 mRNA on the expression of the MDM2 gene.To examine the effect of LINC03074 on mRNA stability, we quantified MDM2 mRNA levels following knockdown of LINC03074 using three different siRNAs, and found a decrease in mRNA levels for all siRNAs (Fig. 3A).In contrast, MDM2 protein levels were increased by LINC03074 knockdown (Fig. 3B).The increase in MDM2 protein by LINC03074 knockdown was rescued by the transient expression of full-length LINC03074 (FL), but not by the ΔAlu mutant (ΔAlu) (Fig. 3B).Next, we verified that LINC03074-induced alterations in MDM2 protein levels were caused by the binding of LINC03074 to MDM2 mRNA.Flag tag fusion MDM2 protein expression plasmids were generated by inserting 3′UTR sequences (FL or Δ5′-Alu) downstream of the CDS of MDM2 (Fig. 3C).These plasmids were transfected together with LINC03074 (FL or ΔAlu) expression plasmids into HEK293 cells, and the protein levels of FLAG-MDM2 were quantified by immunoblotting using an anti-FLAG antibody.In the absence of the 3′UTR, FLAG-MDM2 levels remained unchanged regardless of the presence of LINC03074 (Fig. 3D).However, in the presence of the 3′UTR (FL), FLAG-MDM2 levels were significantly reduced by LINC03074 (FL), but not by LINC03074 (ΔAlu) (Fig. 3D).In the case of Alu elements in the 3′ UTR were absent (Δ5′-Alu), FLAG-MDM2 levels were only slightly reduced by LINC03074 (Fig. 3D).These findings suggest that LINC03074 binding to MDM2 mRNA via Alu elements may influence the post-transcriptional or translational processes of MDM2 gene expression.
LINC03074 enhances STAU1-mediated nuclear export and PKR-induced translational repression of MDM2 mRNA It has been reported that the inverted repeat Alu elements (IRAlus) in the 3′UTR of mRNA serve as a binding site for ADAR1, a dsRNAspecific enzyme that performs A-to-I RNA editing [16].The mRNA with 3′UTR IRAlus edited by ADAR1 is retained in the nucleus through interaction with paraspeckle, which is formed by the nuclear lncRNA, NEAT1, and its binding partner, NONO [22].Alternatively, IRAlus in the 3′UTR of mRNA binds to the dsRNAbinding protein STAU1, which is involved in various RNA metabolic regulations [13].STAU1 binds to IRAlu to facilitate the export of IRAlu mRNA from the nucleus to the cytoplasm, while competitive inhibition of NONO binding to IRAlu prevents NONOmediated mRNA retention in the nucleus [23].We examined the binding of MDM2 mRNA to STAU1, ADAR1, or NONO in the nuclei of TCam-2 cells and the effect of LINC03074 on their binding.RIP assays using nuclear extracts of TCam-2 cells showed that MDM2 mRNA binds to STAU1 and ADAR1, but not NONO (Fig. 4A).Knockdown of LINC03074 suppressed the binding of MDM2 mRNA to STAU1 while increasing its binding to ADAR1 (Fig. 4A).In contrast, LINC03074 bound only to STAU1 but not to ADAR1 and NONO (Fig. 4A).Considering that STAU1 is responsible for RNA shuttling, we investigated whether LINC03074 affected the nuclear export of MDM2 mRNA.MDM2 mRNA was increased in the nucleus and decreased in the cytoplasm following LINC03074 and STAU1 knockdown (Fig. 4B).These results suggested that LINC03074 promotes the recruitment of STAU1 to MDM2 mRNA in the nucleus and facilitates STAU1-mediated nuclear export.
Taken together, the downregulation of LINC03074 increased intracellular MDM2 protein levels, despite decreasing MDM2 mRNA levels in the cytoplasm (Figs.3B and 4B).We speculated that LINC03074-mediated enhancement of STAU1 and MDM2 mRNA interactions in the nucleus leads to the translational repression of MDM2.STAU1 binding to IRAlu mRNA promotes nuclear export and translation [23].However, STAU1-mediated mRNA nuclear export is promoted when the paraspeckle component is downregulated, whereas protein kinase R (PKR)-mediated translational repression in the cytoplasm is promoted [23].PKR is activated by binding to virusderived dsRNA and phosphorylates eukaryotic translation initiation factor 2A (eIF2a), resulting in translational inhibition [24,25].To determine whether PKR can bind to MDM2 mRNA in TGCT cells, we performed RIP assays with cytoplasmic extracts from TCam-2 cells using a PKR antibody.PKR was found to bind to MDM2 mRNA in the cytoplasm, and this interaction was reduced by LINC03074 and STAU1 knockdown (Fig. 4C).Furthermore, STAU1 and MDM2 mRNA binding in the cytoplasm was attenuated by LINC03074 and STAU1 knockdown (Fig. 4C).Finally, we determined whether MDM2 was translationally repressed by PKR activation.MDM2 protein levels were increased by PKR inhibitor treatment of TCam-2 cells (Fig. 4D).The increase in MDM2 protein expression induced by PKR inhibitors was not detected with LINC03074 and STAU1 knockdown (Fig. 4D).These results indicated that LINC03074, similar to paraspeckle components, modulates the nuclear export of STAU1-bound MDM2 mRNA, thereby facilitating PKR-mediated translational repression.

LINC03074 enhances cisplatin-induced apoptosis and cell growth inhibition
To assess whether LINC03074 affects the proliferation of TGCT cells, CCK8 analysis was performed using cisplatin, a platinum chemotherapeutic agent that induces DNA damage in cancer cells by inhibiting DNA repair [26].The growth of TCam-2 cells was inhibited by cisplatin treatment in a concentrationdependent manner (Fig. 5A).LINC03074 knockdown enhanced the growth of TCam-2 cells and attenuated cisplatin-induced inhibition of cell growth (Fig. 5A).In contrast, LINC03074 knockdown had no effect on the growth of non-seminoma-derived NEC8 cells expressing low LINC03074 levels (Supplementary Fig. S3).First, the effect of LINC03074 on the cell cycle was assessed; however, no cell cycle abnormalities due to LINC03074 knockdown or cisplatin treatment were observed under the conditions examined (Supplementary Fig. S4).Next, the effect of LINC03074 on apoptosis was examined using FACS analysis.LINC03074 knockdown reduced the frequency of spontaneous and cisplatininduced apoptosis (Fig. 5B and Supplementary Fig. S5).These results indicate that LINC03074 inhibits the proliferation and promotes the apoptosis of seminoma cells.Furthermore, the responsiveness of seminoma cells to cisplatin-induced DNA damage was enhanced by LINC03074.
LINC03074 increases E2F1 levels and upregulates p73 gene expression MDM2 is a major negative regulator of p53; MDM2 acts as an E3 ubiquitin ligase that recognizes p53 and acts as a transcriptional repressor of p53 [4,5].To test whether LINC03074 affected p53 protein levels and function, we performed immunoblotting using anti-p53 and anti-phosphorylated p53 (Ser15) antibodies.p53 is activated by phosphorylation in response to DNA damage, and its Ser15 residue is the major phosphorylation site [27].Immunoblotting confirmed that cisplatin-induced DNA damage markedly increased the p53 protein levels and promoted p53 phosphorylation (Fig. 6A).Interestingly, LINC03074 knockdown did not affect the p53 or phosphorylated p53 levels (Fig. 6A).MDM2 has been reported to interact with various proteins other than p53, and the apoptosis-related transcription factor E2F1 is one of its target proteins [28,29].Immunoblotting with an anti-E2F1 antibody showed that E2F1 levels were increased by cisplatin treatment and decreased by LINC0374 knockdown, in the presence or absence of cisplatin (Fig. 6A).E2F1 induces apoptosis through several mechanisms, including activation of p53-dependent and -independent pathways and inhibition of survival signaling [30].To elucidate the mechanism by which LINC03074 mediates apoptosis, we examined the effects of LINC03074 knockdown on E2F1 target gene expression.Among the apoptotic genes targeted by E2F1, p73, and BIM are transcriptionally regulated by E2F1, whereas PUMA and NOXA are regulated by E2F1 and p53 [30].Of the four apoptosis-related genes subjected to mRNA quantification, only p73 exhibited decreased mRNA levels following LINC03074 knockdown with cisplatin (Fig. 6B).In addition, p73 mRNA levels increased in response to cisplatin treatment, regardless of LINC03074 knockdown (Fig. 6B).BIM, PUMA, and NOXA mRNAs showed a tendency to increase with LINC03074 knockdown with cisplatin, but no cisplatin addition-dependent increase was observed without LINC03074 knockdown (Fig. 6B).Our results indicate that cisplatin-induced apoptosis of seminoma cells is associated with the increased expression of p73.LINC03074 contributes to the upregulation of p73 by increasing E2F1 expression, which may indirectly affect the expression of other apoptotic genes (Fig. 6C).

DISCUSSION
MDM2 expression levels are associated with chemotherapy resistance in human malignancies and are regulated at multiple levels [8].In this study, we suggest that MDM2 levels are regulated by the testis-specific lncRNA LINC03074 during post-transcription.The LINC03074-MDM2-mediated apoptosis regulatory pathway provides new insights into the mechanisms of the DNA damage response in TGCT cells.A total of 17 pairs of interaction sequences were predicted within the Alu element of LINC03074 with the five Alu elements in the 3′UTR of MDM2 mRNA (data from lncRRIsearch; shown in Fig. 2A).Our results suggest the following: (1) LINC03074 binds complementarily to either Alu element of the MDM2 3′UTR via its own Alu element; (2) LINC03074 modulates the binding of STAU1 and ADAR1 to the IRAlus of the MDM2 3′UTR; and (3) LINC03074 interacts with STAU1 but not ADAR1 in the nucleus.These results suggest that LINC03074 functions as an RNA chaperone for MDM2 mRNA and that LINC03074-induced conformational changes convert the dsRNA-binding factor that binds to the 3′UTR of MDM2.It is speculated that ADAR1 and STAU1 competitively bind to MDM2 mRNA because the interaction of ADAR1 with MDM2 mRNA has been reported to suppress STAU1-MDM2 mRNA The measurements were normalized to control (siControl without PKR inhibitor) protein levels that are indicated at the bottom of each band.
binding [16].Therefore, we concluded that LINC03074 binds to MDM2 mRNA to promote STAU1 recruitment, thereby indirectly suppressing ADAR1-MDM2 mRNA binding.The intracellular molecular ratio of LINC03074 to MDM2 mRNA in the TCam-2 cells was approximately 1:166 (Supplementary Fig. S2).As an example of how a small amount of lncRNA can act on a large number of target molecules, previous reports have shown that SLERT is recycled to induce conformational changes in DDX21, which has approximately 1000 times more molecules than SLERT [31].
Further detailed molecular analysis is needed to determine the mechanism by which LINC03074 exerts its action and modifies more targets than its stoichiometry.In addition, the significance of the differences in LINC03074 levels between carcinoma and normal testis tissues needs to be investigated.
Based on the notion that protein is produced by mRNA translation, total intracellular mRNA decrease and protein increase in MDM2 caused by LINC03074 knockdown seem to be inconsistent (Fig. 3A, B).Besides mRNA to be translated, cytoplasmic dsRNA from viruses has been reported to elicit PKRmediated inhibition of translation initiation [25].PKR is activated by binding to virus-derived dsRNA and phosphorylates eukaryotic translation initiation factor 2A (eIF2A), resulting in translational repression [25].It has been reported that downregulation of paraspeckle components, such as NEAT1 and NONO, increases cytoplasmic 3′UTR IRAlus mRNAs, which increases phosphorylation of PKR and eIF2A, resulting in intracellular translational repression [23].In this study, we demonstrated that downregulation of LINC03074 suppresses STAU1-mediated nuclear export of 3′UTR IRAlus mRNA, resulting in attenuated PKR-mediated translational repression (Fig. 4).LINC03074 had an opposite effect on the posttranscriptional regulation of IRAlus mRNA to the paraspeckle components, but the relationship between LINC03074 and NONO is unclear given that no interaction between them has been found (Fig. 4A).In addition, the mechanism by which cytoplasmic MDM2 mRNA is assigned to the opposite fates-PKR-mediated translational repression and translation initiation-remains unclear.Further investigation of the molecular mechanisms of LINC03074-mediated translational repression will provide new insights into the post-transcriptional regulation of the 3′UTR IRAlus mRNA.
The relationship between MDM2 and p53 expression in TGCTs remains unclear, although MDM2 overexpression plays an important role in suppressing p53 activity in numerous tumors that retain wild-type p53 [32,33].Several reports have suggested that p53 degradation by MDM2 is ineffective in TGCTs.Some studies have shown a positive correlation between MDM2 and wild-type p53 expression levels in TGCTs, while others have shown no correlation [3,34].We found that attenuation of MDM2 levels by LINC03074 led to an increase in E2F1 but not p53 (Fig. 6A).E2F1 is negatively and positively regulated by MDM2 in a p53-independent manner via both direct and indirect mechanisms [28,29].Furthermore, E2F1 plays an important role in regulating cell proliferation and differentiation by significantly influencing cell cycle progression and survival through extensive crosstalk with p53 [35].In this study, we demonstrated that the p73, a target gene of E2F1 related to apoptosis, is involved in the responsiveness of seminoma cells to cisplatin, and its expression is regulated by LINC03074 (Fig. 6B).Considering that the MDM2-E2F1-p73 pathway is predicted to play an important role in chemotherapy resistance of TGCTs, further insights into this pathway may lead to the development of new therapies for TGCTs.Fig. 5 LINC03074 enhances cisplatin-induced apoptosis.A Cell growth assay using TCam-2 cells transfected with siLINC03074 and treated with different concentrations of cisplatin.Absorbance at 450 nm (OD 450 ) was used to estimate cell concentration.Data represent the means ± SEM (n = 3).B Apoptosis assay using LINC03074-knockdown TCam-2 cells treated with cisplatin.TCam-2 cells were transfected with siLINC03074 and treated with 20 μM cisplatin for 48 h.Apoptotic cells were identified by the increase in the fluorescence intensity of FITC-labeled Annexin-V using flow cytometry.Percentage of TCam-2 cells (either with or without siLINC03074) in apoptosis in the presence or absence of cisplatin (n = 3).*P < 0.05.siControl, TCam-2 cells transfected with a negative control siRNA.cytoplasmic fraction.The pellet was washed with the nuclear fractionation buffer and centrifuged at 13000 rpm at 4 °C for 10 min.The pellet was used as the nuclear fraction.The respective markers of the nuclear and cytoplasmic fractions, 5S-rRNA and GAPDH, respectively, were used as controls.

Statistical analysis
Statistical analyses were performed using t-tests or ANOVA, as appropriate.Statistical significance was set at P < 0.05.Each experiment was repeated at least three times.Information on statistical measures is provided in the legend of each figure.

Fig. 2
Fig. 2 Interaction of LINC03074 with MDM2 mRNA.A Schematic drawing of predicted interaction regions between human LINC03074 and MDM2 mRNA.Interaction regions predicted by the lncRRIsearch database (gray dot lines), Alu elements (orange arrows), and inverted Alu pairs (yellow arrows) are shown (upper panel).An example of the predicted base pairs for the regions shown in red and blue in the upper panel is shown (ΔG = −60.91kcal/mol, bottom panel).Sx, Sx1, FLAM_C, Sz and Y, Alu subfamilies; 5′UTR 5′untranslated region, CDS coding sequence, 3′UTR 3′untranslated region.B Enrichment of RNAs by LINC03074 CHART as measured using RT-qPCR.Each enrichment value is shown as a percentage of the measurement for each mock (without C-oligo).Error bars represent +SEM for three qPCR experiments.*P < 0.05.C Schematic representation of full length (FL) and Alu element-deficient (ΔAlu) LINC03074 and biotin-tagged MDM2 3′UTR (FL and Δ5′-Alu).D RNA pull-down assays using the biotin-tagged (bio)-MDM2 3′UTR.In vitro, transcribed bio-MDM2 was incubated with total RNA extracted from HEK293 cells overexpressing LINC03074.RNAs associated with bio-MDM2 3′UTRs were detected via RT-qPCR.Error bars represent +SEM for three qPCR experiments.*P < 0.05.

Fig. 3
Fig.3Reduction of MDM2 protein levels by LINC03074.A MDM2 and LINC03074 RNA levels in LINC03074-knockdown TCam-2 cells measured via RT-qPCR.TCam-2 cells were transfected with siRNAs for LINC03074 (siLINC03074) for 72 h.Data represent the average of three independent measurements normalized to GAPDH mRNA expression.B MDM2 protein levels in LINC03074 knockdown cells transiently expressing the LINC03074 mutant.Western blotting was performed with anti-MDM2 antibody using TCam-2 cells transfected with siLINC03074 for 48 h followed by LINC03074 expression plasmids for 24 h.Band intensity was quantified by Image Lab 6.1.The measurements were normalized to the siControl protein levels that are indicated at the bottom of each band.C Schematic representation of the constructs containing FLAG tagfused MDM2 CDS alone or in combination with 3′UTR.D Western blot analysis using an anti-FLAG antibody against HEK293 cells transfected with FLAG-tagged MDM2 and LINC03074 expression plasmids (left panels).The relative intensity to the band of the control in each first left lane is shown in the bar graph (right panel).

Fig. 4
Fig. 4 Decrease of LINC03074 suppresses STAU1-mediated mRNA nuclear export and PKR-induced translational repression of MDM2.A Effect of LINC03074 on the binding of dsRNA binding proteins and MDM2 mRNA in TCam-2 cell nuclei.RIP assay was performed with each dsRNA binding protein antibody using TCam-2 cell nuclear extracts with LINC03074 knockdown.The level of RNA binding with each dsRNA binding protein was measured via RT-qPCR and is shown as a relative value to the IgG binding level.Error bars represent +SEM for three qPCR experiments.An asterisk above each bar indicates statistical significance for IgG values.*P < 0.05.B Effect of LINC03074 on MDM2 mRNA levels in the nucleus and cytoplasm of TCam-2 cells.The relative expression of MDM2 to 5S-rRNA (nucleus) or GAPDH (cytoplasm) was measured using RT-qPCR.Error bars represent +SEM for three qPCR experiments.An asterisk above each bar indicates statistical significance for siControl values.*P < 0.05.C RIP assay using anti-PKR and anti-STAU1 antibodies with cytoplasmic extracts from TCam-2 with knockdown of LINC03074 or STAU1.Error bars represent +SEM for three qPCR experiments.An asterisk above each bar indicates statistical significance for IgG values.*P < 0.05.D Western blotting of LINC03074 or STAU1 knocked-down TCam-2 cells treated with PKR inhibitor.TCam-2 cells were transfected with siLINC03074 and siSTAU1 for 24 h, followed by treatment with 1μM PKR inhibitor for 24 h.Band intensity was quantified by Image Lab 6.1.The measurements were normalized to control (siControl without PKR inhibitor) protein levels that are indicated at the bottom of each band.

Fig. 6
Fig.6LINC03074 activates E2F1 and p73 pathways.A Western blotting using TCam-2 cells transfected with siLINC03074 for 72 h.Cisplatin was added to the culture medium at a concentration of 10 μM for 72 h before cell extraction.Band intensity was quantified using Image Lab 6.1, and all measurements were normalized to the protein levels of the siControl without cisplatin (indicated at the bottom of each band).B Relative expression levels of apoptotic genes in LINC03074-knockdown cells were measured using RT-qPCR.TCam-2 cells were transfected with siLINC03074 and treated with cisplatin for 48 h (n = 3).*P < 0.05.C Predicted schematic of LINC03074-mediated mechanisms of MDM2 translational repression (left panel) and apoptosis stimulation (right panel).